Cholangiocarcinoma is a highly malignant neoplasm of the biliary tract and its incidence and mortality is rising. Early diagnosis is difficult and currently there is no effective chemoprevention or treatment. Our long-term goal is to define the cellular mechanisms responsible for tumor development and progression in biliary epithelia and to develop new therapies for cholangiocarcinoma. During the previous grant period, we have provided data supporting the important role of cytosolic phospholipase A21 (cPLA21) and cyclooxygenase-2 (COX-2)-derived prostaglandin (PG) signaling in cholangiocarcinoma growth. We have shown that EP1 is a key prostaglandin receptor that importantly contributes to cPLA21/COX-2-induced cholangiocarcinogenesis through transactivation of epidermal growth factor receptor (EGFR). In the current continuation proposal, we hypothesize that transactivation of EGFR by EP1 is crucial for activation of 2-catenin and inducible nitric oxide synthase (iNOS) and that the interactions of these key molecules perpetuate bile duct inflammation and drive carcinogenesis. Consequently, we postulate that simultaneous inhibition of EP1 and EGFR may synergistically disrupt the key steps in cholangiocarcinogenesis and provide effective chemoprevention and treatment. The specific aims of this ongoing investigation are: (1) To evaluate our hypothesis that S-nitrosylation of cPLA21 and COX-2 protein is a novel mechanism by which iNOS regulates cholangiocarcinogenesis; (2) To examine the effect and mechanism for cPLA21/COX-2/PGE2-induced 2-catenin activation in human cholangiocarcinoma cells; and (3) To examine the role of EP1-mediated EGFR transactivation in iNOS expression and to evaluate the effect of concomitantly inhibiting EP1 and EGFR on cholangiocarcinoma growth. Complementary approaches of cultured cholangiocarcinoma cells and animal models will be employed. The proposed research is highly significant since it will investigate important signaling pathways during cholangiocarcinogenesis and evaluate novel targeted therapy for chemoprevention and treatment.